副鸡禽杆菌外膜囊泡的生物学活性分析

Biological Activity Analysis of Avibacterium paragallinarum Outer Membrane Vesicles

为研究副鸡禽杆菌(Apg)外膜囊泡(OMVs)的生物活性,提取了A型副鸡禽杆菌的外膜囊泡。纯化后的OMVs用透射电镜观察其形态结构;将OMVs加入到鸡巨噬细胞(HD-11)培养液中,检测一氧化氮(NO)产生量的变化;用荧光定量PCR(qPCR)检测HD-11各炎症相关因子的表达量变化情况;将OMVs经肌肉注射接种SPF鸡,检测鸡血清IgG抗体产生情况,并用3种血清型的副鸡禽杆菌强毒菌株攻击免疫鸡,测定其免疫保护率。结果显示,电镜下的副鸡禽杆菌OMVs呈球形,直径在20~300 nm;OMVs可极显著提高HD-11中NO产量;qPCR结果显示,OMVs作用可显著上调鸡巨噬细胞IL-2、IL-6、 IL-1β、IL-10、iNOs等炎症相关因子的表达。免疫鸡可以产生特异性的IgG抗体。用A型副鸡禽杆菌攻毒,免疫保护率为70%,对异种血清型交叉保护性较弱,仅有10%~30%。本研究为深入研究副鸡禽杆菌外膜囊泡的功能奠定了基础。

In order to study the biological activity of the outer membrane vesicles(OMVs) of Avibacterium paragallinarum(Apg), the OMVs were extracted, its morphological structure was observed, and its biological activity as detected in this experiment. The production changes of nitric oxide(NO) and some inflammation factor expression related to OMVs were detected after the OMVs were added to chicken macrophage(HD-11) culture medium. Serum IgG antibodies were measured after intramuscular injection of SPF chicken with OMVs. The injected chickens were challenged by Apg strains, and its immune protection was detected finally. The results showed that the OMVs of Apg were spherical with a diameter of 20~300 nm under transmission electron microscopy. The NO yield in HD-11 was significantly increased by OMVs. qPCR results showed that the OMVs significantly up-regulated the expressions of inflammatory related factors, such as IL-2, IL-6, IL-1β, IL-10 and iNOs in chicken macrophages. Vaccinated chickens produced specific IgG and nasal mucosa sIgA antibodies against Apg, which provided a protection rate of 70% against the challenging of A serotype Apg. However, the cross-protective against the heterologous serotype challenge was weaker, only 10% to 30%. This study lays a foundation for further exploring the function of the outer membrane vesicles of Avibacterium paragallinarum.